A. Field of the Invention
The present invention relates generally to the fields of biochemistry, molecular biology, and virology. More particularly, it relates to the identification of 259 nucleotides of previously unrecognized sequence located at the 3′ end of the GB virus B (GBV-B) genome.
B. Description of Related Art
Chronic hepatitis C is a major threat to the public health. Serologic surveys suggest that as many as 3.9 million Americans are chronically infected with the responsible virus, hepatitis C virus (HCV) (Alter, 1997). These individuals are at increased risk of developing progressive hepatic fibrosis leading to cirrhosis and loss of hepatocellular function, as well as hepatocellular carcinoma. The course of chronic hepatitis C is typically lengthy, often extending over decades, with insidious clinical progression usually occurring in the absence of symptoms. Nonetheless, liver disease due to HCV results in the death of 8,000–10,000 Americans annually, and chronic hepatitis C is the most common cause of liver transplantation within the U.S.
Therefore, HCV is a major public health problem. However, therapy for chronic hepatitis C is problematic. Recombinant interferon-α is approved for treatment of chronic hepatitis C (Consensus Development Panel, 1997). The benefit of interferon-α results primarily from its antiviral properties and its ability to inhibit production of virus by infected hepatocytes (Neumann et al., 1998). Nonetheless, even under optimal therapeutic regimen, the majority of patients with chronic, hepatitis C fail to eliminate the virus or resolve their liver disease. Treatment failures are especially common in persons infected with genotype 1 HCV, unfortunately the most prevalent genotype in the U.S. Thus, there is an urgent need to better understand the virus and develop better treatment. Unfortunately, technical difficulties in working with HCV have made it necessary to use infectious surrogate viruses in efforts to develop treatments and vaccines for HCV.
Scientists' efforts to better understand HCV and to develop new drugs for treatment of hepatitis C have been stymied by two overwhelming technical deficiencies: first, the nonexistence of a high permissive cell line that supports replication of the virus and second, the absence of a permissive animal species other than chimpanzees, which are endangered and therefore available on a limited basis.
Presently, those who are working on HCV treatment and prevention are employing an infectious chimeric virus of sindbis and HCV and/or an infectious clone of pestiviruses as surrogate virus models in HCV drug discovery efforts, due to the above technical difficulties of working with HCV. Alternatively, they are using isolated proteins or RNA segments of HCV for biochemical and structural studies. This approach precludes functional studies of virus replication and its inhibition.
GBV-B is a hepatotropic flavivirus that has a unique phylogenetic relationship to human HCV and strong potential to serve as a surrogate virus in drug discovery efforts related to hepatitis C antiviral drug development. GBV-B causes acute hepatitis in experimentally infected tamarins (Simons et al., 1995; Schlauder et al., 1995; Karayiannis et al., 1989) and can serve as a surrogate virus for HCV in drug discovery efforts (due to technical difficulties in working with HCV). GBV-B virus is much closer in sequence and biological properties than the above-described models. It will be easier to make biologically relevant chimeras between HCV and GBV-B than by using more distantly related viruses. GBV-B is hepatotropic (as is HCV), whereas the viruses used in these competing technologies are not. In view of the above, an infectious clone of GBV-B would be useful to those working on HCV treatment and prevention.
Unfortunately, the use of GBV-B as a surrogate or model for HCV has not been possible in the past, because no infectious molecular clone of GBV-B virus genome could be prepared. It is now known that this obstacle was encountered because the GBV-B genome was believed to be 259 nucleotides shorter than its actual length (Muerhoff et al., 1995; Simons et al., 1995). Others, previous to the inventors, had failed to realize that the 3′ sequence of GBV-B was missing from the prior sequences. Without this 3′ sequence, it is not possible to prepare an infectious GBV-B molecular clone.